The present invention relates to a method for culturing living cells and, more particularly, to a method for economically culturing living cells in vitro in a manner which closely simulates the in vivo environment in which the living cells normally exist.
There is a large array of bio-medically useful products which are derived from mammalian systems. These products, arbitrarily classified according to their increasing size and molecular complexity, include (a) small molecules such as peptides (anti-diuretic hormone, oxytocin, antigenic portion of viral proteins); (b) intermediate molecules (insulin, interferon, lymphokines, growth hormone, thymus stimulating factor); (c) large complex molecules such as antibodies; and (d) whole organisms and cells (antibody producing cells, tumor cells, endothelial cells, vaccines).
Modern methods of manufacturing natural products include chemical synthesis, genetic engineering and mass cell culture. At the present time, chemical synthesis is not often used for it is only applicable to simple, small molecules such as peptides with 20 amino acids or less, and even as to these the process is very expensive. Genetic engineering is not applicable for making large complex molecules such as natural antibodies to be used for treatment and other purposes. Moreover, both with chemical synthesis and with genetic engineering, each new product is a major research and manufacturing endeavor and, hence, these methods are not easily applicable to making families of molecules such as antibodies or lymphokines.
In contrast, all natural products can, in principle, be produced by the mass culture of the very organisms or cells making such products. Once a suitable culture system has been established, moreover, it can be generally applied to all products produced by cells or organisms without the need for embarking upon a major research effort for each different product.
Despite these recognized advantages of mass culture as a potentially universal means for producing the large array of products obtainable from mammalian systems, the use thereof has not heretofore been practical, primarily for reasons of economics, i.e., the cost of culturing a given number of cells or organisms is extremely high.
The system in which cells or organisms are cultured in vitro theoretically is designed to provide an environment in which the cells or organisms can perform the essential functions (e.g., growth, product production, waste excretion) performed by the cells or organisms in vivo. In practice, however, the natural and/or synthetic culture media conventionally employed for this purpose are far from ideal in many respects and, more importantly, are extraordinarily expensive. In addition, known culture systems are not easily adapted to accommodate the large number of cells required to economically produce a sufficient quantity of a given product, and lack means for economically and efficiently removing from the system the desired products being made by the culturing cells.
One of the methods known for the in vitro culture of a large number of cells is "suspension culture", wherein a suspension of cells and culture medium is brought about and sustained by stirring or movement of the apparatus containing the cells and medium. Suspension culture is desirable since the environment around every cell is substantially the same and each cell can derive its nutrition from, and excrete its waste and products into, the medium surrounding the entire surface of the cell. However,.the suspension culture processes practiced in the art, wherein culture medium is caused to flow in and out of an apparatus (culture chamber) containing the cell/culture suspension exhibit a number of substantial disadvantages, particularly with respect to the means, e.g., filters, employed for retaining the cells in the culture chamber while medium flows therethrough and the ability to collect from the system the desired products being made by the cells.
Apart from the disadvantages of typical suspension culture techniques, it is also known that some cells simply will not grow, or grow only poorly when in suspension. In these cases, it has been proposed to attach the cells to a surface or substrate thereby facilitating their ability to grow in standard tissue culture media. The substrate may be the surface of a culture chamber or vessel, micro-beads (see, e.g., M. Hirtzenstein, et al., "Microcarriers For Animal Culture: A Brief Review Of Theory And Practice", Proceedings Of The Third General Meeting Of The European Society For Animal Cell Technology (1979)) or the surface of hollow fibers (see, e.g., R. A. Knazek, "Cell Culture Of Artificial Capillaries: An Approach To Tissue Growth In Vitro", Science, 178, p. 65 (1972)).
Significant advances in the art of in vitro culture of cells are described in my U.S. Pat. Nos. 3,964,467 and 4,064,006 with respect both to the medium in which the cells are cultured and to apparatus which facilitate the mass culture of cells either in suspension or affixed to a surface. In particular, the foregoing patents describe the use of fresh flowing cell-free lymph as a culture medium for living cells. The cells are contacted with the flowing cell-free lymph in a suitable chamber in which the cells may exist either in a free state or bound to the surface of the vessel or to inert carrier particles. In one embodiment, the cell-free lymph and the cells to be cultured are contacted in a centrifuge which is periodically accelerated and decelerated to, respectively, hold the cells in a fixed position and then to disturb the cells from this fixed position and disperse them in fresh cell-free lymph.
As described in the aforementioned U.S. Patents, the procedure and apparatus developed for in vitro culture also makes possible an "in vivo - in vitro" culture method wherein the host is linked, by way of its lymph, to explant tissues or cells. Thus, a patient or subject is treated, by performing thereon a thoracic duct fistula (and optionally by raising the patient's capillary filtration flow) so as to assure that the tissue through which the lymph percolates does not significantly add or subtract molecules to or from the lymph. (See generally my U.S. Pat. Nos. 3,719,182 and 3,857,393 regarding this procedure). Lymph collected from the patient in this manner is treated so as to separate cells therefrom, and the cell-free lymph obtained in this manner is then utilized as the culture medium for living cells.
The processes described in the present invention represent important advances over my earlier culture methods, particularly with respect to the ability to provide an in vitro culture system wherein the cells are subjected to an environment which permits them to function in a manner closely simulating that encountered in vivo, permits the efficient removal of desired products from the cells being cultured and provides important advantages in the efficiency, economy and flexibility of operation.